The genome of flax (Linum usitatissimum) assembledde novo from short shotgun sequence reads

Zhiwen Wang1, Neil Hobson2, Leonardo Galindo2, Shilin Zhu1, Daihu Shi1, Joshua McDill2, Linfeng Yang1, Simon Hawkins3,

Godfrey Neutelings3, Raju Datla4, Georgina Lambert5, David W. Galbraith5, Christopher J. Grassa6, Armando Geraldes6,

Quentin C. Cronk6, Christopher Cullis7, Prasanta K. Dash8, Polumetla A. Kumar8, Sylvie Cloutier9,10, Andrew G. Sharpe4,

Gane K.-S. Wong1,2,11,*, Jun Wang1,12,13,* and Michael K. Deyholos2,*

1BGI-Shenzen, Bei Shan Industrial Zone, Yantian District, Shenzhen 518083, China,2Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada,3Universite Lille-Nord de France, Lille 1 Unite Mixte de Recherche Institut National de la Recherche Agronomique 1281, Stress

Abiotiques et Differenciation des Vegetaux, F-59650 Villeneuve d’Ascq Cedex, France,4National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, Saskatchewan, S7N 0W9, Canada,5University of Arizona, School of Plant Sciences and BIO5 Institute, Tucson, AZ 85721, USA,6Department of Botany, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada,7Case Western Reserve University, Cleveland, OH 44106, USA,8National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, Pusa Campus, New Delhi 110012, India,9Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M1, Canada,10Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada,11Department of Medicine, University of Alberta, Edmonton, Alberta, T6G 2E1, Canada,12The Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark, and13Department of Biology, University of Copenhagen, Denmark

Received 28 June 2011; revised 25 May 2012; accepted 26 June 2012; published online 14 August 2012.

*For correspondence (e-mails deyholos@ualberta.ca, wangj@genomics.org.cn and gane@ualberta.ca).

SUMMARY

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally

relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of

the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced

using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approx-

imately 94· raw, approximately 69· filtered) short-sequence reads (44–100 bp), produced a set of scaffolds

with N50 = 694 kb, including contigs with N50 = 20.1 kb. The contig assembly contained 302 Mb of non-

redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs

aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs

and fosmids showed some mis-assembly of regions at the genome scale. A total of 43 384 protein-coding

genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86%

of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene

level. Analysis of the synonymous substitution rates (Ks) observed within duplicate gene pairs was consistent

with a recent (5–9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed

enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop,

including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-

genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence

information for some plant species.

Keywords: whole-genome shotgun, DNA sequencing, Illumina, flax, Malpighiales, industrial crops.

INTRODUCTION

Flax (Linum usitatissimum) has been used by humans for at

least nine millennia and possibly for as long as 30 millennia

(van Zeist and Bakker-Heeres, 1975; Zohary and Hopf, 2000;

Kvavadze et al., 2009). Varieties of this species are now

cultivated for either fibers or seed, on approximately 3 Mha

in over 20 countries (http://faostat.fao.org/). The bast fibers

ª 2012 The Authors 461The Plant Journal ª 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 72, 461–473 doi: 10.1111/j.1365-313X.2012.05093.x

that grow in the outer layers of the flax stem are made up of

remarkably long cells that are rich in crystalline cellulose and

have a very high tensile strength (Mohanty et al., 2000).

These fibers are the source of linen textiles, and their use in

composite materials is an area of active research (Bodros

et al., 2007). The seed of flax (i.e. linseed) produces oil that is

rich in unsaturated fatty acids, especially a-linolenic acid

(C18:3), polymers of which are used in linoleum, paints and

other finishes. Consumption of the oil or seed has been

reported to have beneficial effects on cardiovascular health

and in the treatment of certain cancers and inflammatory

diseases (Singh et al., 2011). Health benefits are derived

from both the a-linolenic acid and other components of the

seed, including lignans such as secoisolariciresinol dig-

lucoside (SDG), which is an antioxidant and the precursor of

several phytoestrogens (Toure and Xu, 2010). Flax seed is

also used in animal feed to increase levels of a-linolenic acid

in meat or eggs (Simmons et al., 2011). In total, flax is a

source of at least three classes of commercial products: fiber,

seed oil, and nutraceuticals. Increased understanding of the

genes affecting the quality and yield of these bioproducts will

contribute to crop improvement for flax and other oil- or fiber-

producing species.

In addition to its economic uses, flax has several charac-

teristics that make it an interesting subject for scientific

inquiry. Among these are the diversity of the flax family and

its relatives. The genus Linum comprises an estimated 180

species distributed over six continents (McDill et al., 2009).

The genus displays interesting variation in evolutionarily

significant characteristics such as breeding system (disty-

lous and homostylous species are known), flower color

(ranging from white to yellow, red and blue), pollen

morphology (tricolpate or multiporate) and edaphic ende-

mism (i.e. restriction to specific soil types). The family

Linaceae has impressive ecological and morphological

diversity, with more than 270 species in 14 genera ranging

from tiny, serpentine-endemic annuals in the California

chaparral (the genus Hesperolinon) to canopy trees in South

American black-water forests (Hebepetalum) and tendrillate

vines in Indonesian jungles (Indorouchera). Linum and the

Linaceae have recently been the subject of molecular

phylogenetic investigations using chloroplast markers, the

nuclear ribosomal internal transcribed spacer (ITS), and

retrotransposon sequences (McDill et al., 2009; Fu and

Allaby, 2010; McDill and Simpson, 2011; Smykal et al.,

2011). The Linaceae family belongs to the order Malpighi-

ales, of which three species have been fully sequenced:

Populus trichocarpa (black cottonwood, Salicaceae), Ricinus

communis (castor bean, Euphorbiaceae) and Manihot escul-

enta (cassava, Euphorbiaceae) (Tuskan et al., 2006; Chan

et al., 2010; http://phytozome.net. The phylogenetic relation-

ships among families in the Malpighiales are not entirely

resolved (Wurdack and Davis, 2009), but recent estimates

suggest that the Linaceae diverged as a lineage distinct from

other families of Malpighiales approximately 100 MYA

(Davis et al., 2005). The lineage leading to Arabidopsis

thaliana, whose genome is commonly used in comparative

genome analyses, is thought to have diverged from the

lineage leading to Malpighiales between 100 and 120 MYA

(Tuskan et al., 2006).

Flax is also of general scientific interest because some

varieties of flax (e.g. Stormont Cirrus) exhibit rapid changes

in nuclear DNA content (Cullis, 1981b, 2005). Differences in

nuclear C-value of up to 15% have been reported among

first-generation progeny of self-pollinated individuals that

were subjected to specific temperature or fertilizer regimes

(Evans et al., 1966). Altered copy numbers of many types of

genetic elements contribute to the observed changes in

genome size, including rRNA genes and an unusual inser-

tional element named LIS-1 (Goldsbrough et al., 1981; Chen,

1999). Increased genomic sequence information for flax may

help to further elucidate the basis of genome plasticity.

Patterns of genetic inheritance in L. usitatissimum are

typical of a diploid. L. usitatissimum has n = 15 chromo-

somes, which is common in certain lineages of the genus.

Other species of Linum have been reported to have n = 8, 9,

10, 13, 14, 18 or 27 chromosomes (Rogers, 1982), consistent

with a history of repeated genome duplications (polyploidy)

and possible instances of aneuploid reductions or increases.

Published estimates of the nuclear DNA content of flax range

from C = 538 Mb to C = 685 Mb, although reassociation

kinetics analyses have produced estimates as low as

C = 350 Mb (Evans et al., 1972; Cullis, 1981a; Marie and

Brown, 1993). The reassociation kinetics studies also show

that approximately 50% of the genome is composed of

low-copy-number sequences, with 35% highly repetitive

sequences, and the remaining approximately 15% in the

middle-repetitive fraction, which typically represents trans-

posable elements (Cullis, 1980). Genomic sequence

resources for L. usitatissimum in public databases include

286 294 ESTs and 80 339 BAC end sequences in GenBank

(http://www.ncbi.nlm.nih.gov/nucleotide/). The majority of

these sequences were obtained from the cultivar CDC

Bethune (Venglat et al., 2011). A linkage map based on

SSRs (Cloutier et al., 2009) and a 368 Mb BAC physical map

of CDC Bethune (Ragupathy et al., 2011) have been pub-

lished recently. Microarray gene expression and proteome

studies of flax have also been reported (Lynch and Conery,

2000; Roach and Deyholos, 2007; Fenart et al., 2010).

To further increase the available sequence resources for

flax, we have performed whole-genome sequencing of the

CDC Bethune cultivar of Linum usitatissimum, which is a

highly inbred, elite linseed cultivar grown on the majority of

flax acreage in Canada (Rowland et al., 2002). CDC Bethune

does not exhibit any of the phenotypic plasticity that

accompanies the rapid genome change observed in Stor-

mont Cirrus. We used a whole-genome shotgun approach to

sequence CDC Bethune, using de novo assembly of exclusively

462 Zhiwen Wang et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

short reads, a strategy that has previously proven effective in

vertebrates (Li et al., 2010) and date palm (Phoenix dactylif-

era) (Al-Dous et al., 2011). The primary objective was to

characterize the gene space of flax, in support of gene

discovery, marker development and reverse genetics activ-

ities that are already underway. The analysis of the WGS

assembly is presented here.

RESULTS

To obtain a whole-genome shotgun (WGS) assembly of flax,

we extracted DNA from axenically grown, etiolated seedlings

of a linseed cultivar of L. usitatissimum, CDC Bethune, to

produce size-selected sequencing libraries based on five

insert sizes ranging from 300 bp to 10 kb in length (Table 1).

Each library was used as a template in paired-end or mate-

pair sequencing reactions in an Illumina GAII genome

analyzer, producing a total of 34.98 Gb of reads that ranged

between 44 and 100 bp in length. After filtering low-quality

sequences, the remaining 25.88 Gb were used in assembly.

Using flow cytometry, we estimated the nuclear DNA content

of CDC Bethune to be 2N = 2C = 0.764 pg (r�x ¼ 0:07 pg),

corresponding to a haploid nuclear genome of 373 Mb. The

sum of the WGS sequence reads obtained therefore repre-

sented approximately 94· coverage (raw reads) and 69·coverage (filtered reads) of the nuclear genome of flax.

SOAPdenovo is a de Bruijn graph-based assembly pro-

gram designed to use short WGS reads as its input (Li et al.,

2009). Applying this software to filtered Illumina reads, we

generated a de novo assembly in which 50% of the assembly

(N50) was contained in 4427 contigs of 20 125 bp or larger

(Table 2). All contigs were further assembled into 88 384

scaffolds (‡100 bp), of which 132 scaffolds containing 50%

of the assembly were 693.5 kb or larger (N50 = 693.5 kb).

The contigs and scaffolds each contained a total of 302 and

318 Mb of unique sequence, respectively. Therefore, as a

proportion of the nuclear DNA content estimated by flow

cytometry, the WGS assembly contained an equivalent of

81% genome coverage in contigs and 85% genome coverage

in scaffolds. The modal sequencing depth in the assembly

was 45· (Figure S1). The distribution of assembly unit

lengths is shown in Figure 1(a). The longest contig in the

assembly was 151.8 kb and the longest scaffold was

3.09 Mb. Gaps within scaffolds ranged in length from 1 to

10 807 bp, with a median length of 148 bp (Figure 1b). The

WGS contigs contained 40% G+C bases, which is higher

than most other sequenced dicot genomes (Figure 1c). The

same G+C proportion was observed in an analysis of 80 340

capillary-sequenced BAC end sequences from the same

cultivar of flax, thereby confirming that the observed G+C

bias is not merely an artifact of the sequencing technology

used (Ragupathy et al., 2011).

To assess the accuracy of the de novo assembly, we

compared it to multiple independent sources of flax DNA

sequence information. First, we obtained the complete set

of 286 852 Linum usitatissimum ESTs from the National

Center for Biotechnology Information (NCBI). Most of these

accessions (93%) were from the same cultivar (CDC Beth-

une) that was used for WGS sequencing. Using BLAT

software (Kent, 2002), we found that 248 927 of the

286 852 NCBI ESTs (87%) aligned to at least one WGS

scaffold, with ‡95% coverage of the EST length and ‡95%

sequence identity (Table 3). When ESTs were filtered by

length (removing some low-quality sequences), we

observed that at least 93% (217 875/234 547) of the NCBI

flax ESTs with a length ‡400 bp aligned to the WGS

scaffolds. We also aligned the WGS scaffolds with fosmids

and BACs that had been sequenced using capillary or

pyrosequencing methods, and that represented a total of

1.3 Mb of non-redundant genomic sequence (Table 4 and

Figure S2). Within aligned blocks, sequence identity was

typically ‡99%. However, globally, only between 81.4% and

99.2% of BAC or fosmid sequences aligned with the WGS

scaffolds. Moreover, three BACs aligned best to non-

contiguous segments of different scaffolds, indicating that

the long-range continuity of the WGS assembly was limited

in its accuracy. We further characterized the long-range

accuracy of the WGS assembly by comparing the WGS

scaffolds to 80 340 previously described BAC end

sequences (BES) obtained from paired, Sanger dideoxy

sequencing reads of two CDC Bethune BAC libraries

Table 1 Summary statistics of the paired-end libraries used in WGSsequencing

Insertsize

Readlength

Totaldata (Gb)

Filtereddata (Gb)

Sequencedepth (·)

300 bp 44/75/100 12.87 9.81 26.3500 bp 44/75/100 15.41 10.54 28.32 kb 44 3.19 2.77 7.45 kb 44 1.82 1.61 4.310 kb 44 1.69 1.15 3.1Total 34.98 25.88 69.4

Table 2 Statistics for the WGS assembly

Contig Scaffold

Length (bp) Number Length (bp) Number

N90 2487 17 966 82 101 601N80 7548 11 535 225 440 372N70 11 682 8349 349 810 261N60 15 741 6126 497 164 186N50 20 125 4427 693 492 132Longest 151 807 – 3 087 368 –Total size 302 170 579 – 318 247 816 –Total number(‡100 bp)

– 116 602 – 88 384

Total number(‡2 kb)

– 19 160 – 1458

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ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

(Figure 1d) (Ragupathy et al., 2011). Of the 40 099 BES

accessions that aligned to masked WGS scaffolds with high

stringency (‡99% sequence identity, ‡400 bp alignment

length and ‡95% BES coverage), 9668 were pairs (i.e. from

opposite ends of the same BAC) in which both members

aligned to the same WGS scaffold. In each case, the length

(a) (b)

(c) (d)

Figure 1. Genome assembly results.

(a) Assembly units (i.e. contigs or scaffolds) were

sorted by descending length, and the cumulative

DNA content was plotted as a function of the

total number of assembly units. The total

genome size of 373 Mb (measured by flow

cytometry) is represented by a dashed line, and

the N50 and N90 assembly units are indicated by

filled and open circles, respectively.

(b) Distribution of gap length within the scaffold

assembly.

(c) G+C content as a proportion of total bases

measured in 500 bp bins throughout the flax

(Lus) WGS assembly, compared to genomes of

nine other fully sequenced dicots: Arabidopsis

thaliana, Cucumis sativa, Glycine max, Ricinus

communis, Populus trichocarpa, Malus domes-

tica, Manihot esculentum, Medicago truncatula

and Vitis vinifera.

(d) Distribution of the length of intervening

sequence in scaffold/BES alignments. The length

of scaffold DNA between paired BES that aligned

to the same scaffold was calculated, and is

plotted as a frequency distribution for each of

the pairs of aligned BES.

Table 3 Alignment of capillary-sequenced ESTs to WGS scaffolds

EST coveragein alignment (%)

Alignmentidentity (%)

EST length(bp)

Aligned ESTs(number)

Total ESTs queried(number)

ESTsaligned (%)

‡90 ‡95 >0 262 748 286 852 91.6‡95 ‡95 >0 248 927 286 852 86.8‡90 ‡95 ‡400 225 579 234 547 96.2‡95 ‡95 ‡400 217 875 234 547 92.9

Table 4 Alignment of capillary-sequenced BACs and fosmids to WGS scaffolds

BAC/fosmidBAC/fosmidsize (kb)

Matchingscaffold

Scaffoldsize (kb)

Alignment(kb)

Identity(%)

Gaps(%)

JX174446 214.6 156 2281.8 214.6 95.6 3.1RL10-contig00001 190.5 107 560 190.5 97.7 1.5JX174448 184.9 28 1933.6 119.2 98 1.4

23 781.8 64.2 99.2 0.6JX174447 180.1 1036 248.3 23.5 94 2.5

33 2127.3 156.5 94.9 3.3JX174445 179.1 155 809.2 126 95.4 2.2

177 934 53.1 95 1.7JX174444 172.5 96 1083.7 172.5 91.8 5.3JX174449 130.3 931 463.5 130.3 93.9 5.2HQ902252 39.8 1486 346.5 39.8 91.2 7.7JN133299 34.6 465 878.6 34.6 81.4 2.1JN133300 31.5 898 1045 31.5 98.6 1.1JN133301 26.2 1856 140.6 26.2 86.7 6.2

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of scaffold region between the aligned BES pairs was

calculated, and the distribution of these lengths is plotted in

Figure 1(d). The median length of scaffold sequence was

133.4 kb, and the mean length was 140 kb. These results

are consistent with the 135 and 150 kb mean insert lengths

reported for the two original CDC Bethune BAC libraries.

However, a minority of alignments (1448/9668; 15%) had

intervening scaffold sequence lengths that deviated greatly

from the mean (<100 kb or >200 kb), indicating that addi-

tional studies are required to improve the long-range

accuracy of the assembly.

Repetitive elements

To identify various types of middle-repetitive DNA

sequences within the CDC Bethune WGS assembly, the

complete set of WGS scaffolds was submitted to REPEAT-

MASKER version 3.3.0 (http://www.repeatmasker.org/), which

identified 15.8 Mb of sequence (5.2% of a total of 302 Mb

WGS contigs) as having significant similarity to the subjects

in RepBase (http://www.girinst.org/repbase/). The set of

repeats identified by this homology-based analysis was

expanded substantially by application of de novo repeat

identification methods, resulting in a total of 73.8 Mb (24.4%

of WGS contig assembly) being annotated as sequence with

similarity to mobile elements (Table 5). As expected,

retroelements were the most common mobile elements

found in the genome (20.6% of WGS contigs), and these

were represented primarily by LTR type retroelements

(18.4%) followed by long interspersed elements (LINEs)

(2.2%). All DNA transposons represented a much smaller

proportion of the WGS contig sequences (3.8%), and most

were Mutator-type elements (2.1% of WGS contig

sequence). The proportions of the various types of mobile

elements are similar to what has been reported in other full-

sequenced dicot genomes of comparable size. The unusual

5.8kb LIS-1 insertion sequence (GenBank accession AF104351)

that has been described in other varieties of flax was not

found in its entirety in the CDC Bethune assembly; the larg-

est LIS-1 fragment detected was 543 bp (Chen et al., 2005).

Non-coding RNAs

Non-coding RNAs comprise the majority of cellular RNAs

and play an important role in translation (tRNA and rRNA),

synthesis of the translational apparatus (snRNA), and gene

regulation (miRNA). We searched the WGS assembly for

sequences characteristic of these four types of non-coding

Table 5 Transposable elements identified in the flax WGS assembly

Class Order SuperfamilyNumber ofelements

Elementspercentage (%)

Sequenceoccupied (bp)

Sequence percentageof transposableelements (%)

Sequencepercentage ofgenome (%)

Retrotransposons LTR Copia 89 951 38.31 29 594 882 40.08 9.79Gypsy 72 626 30.93 25 123 127 34.02 8.31Unclassified 2797 1.19 902 298 1.22 0.30

DIRS DIRS 2 0.00 102 0.00 0.00PLE Penelope 548 0.23 30 214 0.04 0.01LINE RTE 11 0.00 618 0.00 0.00

L1 27 632 11.77 6 684 243 9.05 2.21SINE Unclassified 1 0.00 49 0.00 0.00

DNA transposons TIR Tc1-Mariner 191 0.08 38 231 0.05 0.01hAT 7935 3.38 1 986 522 2.69 0.66Mutator 21 124 9.00 6 320 424 8.56 2.09P 2 0.00 96 0.00 0.00Harbinger 1384 0.59 344 876 0.47 0.11En-Spm/CACTA 8330 3.55 2 372 592 3.21 0.79

Helitron Helitron 2154 0.92 434 859 0.59 0.14Unclassified Unclassified 95 0.04 8981 0.01 0.00

Total 234 783 100.00 7 384 2114 100.00 24.29

LTR, Long Terminal Repeat; DIRS, Dictyostelium Intermediate Repeat Sequence; PLE, Penelope; LINE, Long Interspersed Nuclear Element; SINE,Short Interspersed Nuclear Element; TIR, Terminal Inverted Repeat.

Table 6 Non-coding RNA species identified in the flax WGSassembly

Type Sub-typeCopynumber

Meanlength(bp)

Totallength(bp)

Percentageof genome

miRNA 297 120.49 35 785 0.01tRNA 1100 74.75 82 224 0.03rRNA 1100 90.99 100 088 0.03

18S 65 186.97 18 653 0.0028S 14 121.43 1700 0.005.8S 11 123.18 1355 0.005S 1010 77.60 78 380 0.02

snRNA 462 120.05 55 462 0.02CD box 264 102.57 27 079 0.01HACA box 41 119.85 4914 0.00Splicing 157 149.48 23 469 0.01

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ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

RNAs (Table 6) (Griffiths-Jones et al., 2003; Nawrocki et al.,

2009). At least 297 putative miRNA precursor loci with

similarity to known miRNAs were identified (Table S1), and

more than 1000 copies were found of both tRNAs and 5S

RNAs. These frequencies are similar to what has been

reported in other species of comparable genome size.

However, an unexpectedly low number of 45S RNAs were

identified, with as few as 11 copies of the 5.8S RNA com-

ponent of the 45S locus found in the WGS assembly.

Plastid sequences

We searched the WGS assembly for fragments with similarity

to plastid DNA. We identified 31 scaffolds that consisted pri-

marily of chloroplast DNA, but we did not detect a complete

assembly of the plastid genome among the flax WGS scaf-

folds. However, many occurrences of plastid-derived

sequences were observed within the scaffolds of nuclear

DNA. Using BLASTN (e-value £10)4 or lower) (Altschul et al.,

1997), we compared the WGS assembly to a draft of the

L. usitatissimum chloroplast genome that was indepen-

dently sequenced (J. McDill, unpublished results). This

search revealed 1356 regions with significant similarity to flax

chloroplast DNA, ranging from 23 to 5899 bp in length in 416

scaffolds. In total, these regions represented an approxi-

mately 89% coverage of the draft flax chloroplast genome

(including only a single copy of the chloroplast inverted

repeat region). The size distribution of these regions con-

forms to the distributions of NUPT (nuclear plastid DNA)

observed in otherspecies (Figure S3) (Richly andLeister,2004).

Gene prediction

To predict the locations of protein-coding genes within the

repeat-masked WGS assembly, we used two de novo hidden

Markov model-based gene-finding programs: Glimmer-

HMM and Augustus (Majoros et al., 2004; Stanke et al.,

2008). The output of these programs was integrated with

WGS alignments of flax ESTs and other empirically estab-

lished plant transcript sequences to produce a consensus

gene set using GLEAN (Elsik et al., 2007). The consensus gene

set contained a total of 43 484 genes, with one transcript

model per gene. The mean and median gene lengths [coding

sequences (CDS), no introns] of the consensus gene set

were 1200 and 987 bp, respectively. A total of 40 971 genes

had a predicted length ‡300 bp. The distributions of CDS,

exon and intron lengths are shown in Figure 2 for the pre-

dicted flax genes and genes from five other representative

genomes.

To assess the coverage and accuracy of the gene predic-

tion, we compared the filtered set of 43 484 predicted flax

genes (which excluded most transposable elements) to the

complete set of flax ESTs from the NCBI databases (Table 7).

We aligned the CDS regions of the predicted genes to the

286 852 NCBI EST sequences using BLASTN. We observed

that 248 210/286 852 NCBI flax ESTs (86.5%) aligned to one

or more WGS CDS (BLASTN e-value £10)20). Because many

ESTs potentially included UTRs, which are not part of CDS

datasets, we repeated the comparison, using only the

239 220 NCBI EST accessions that were ‡400 bp long. Of

the length-filtered NCBI flax ESTs, 222 846/239 220 (93.2%)

aligned to one or more CDS from the predicted flax WGS

proteome (BLASTN e-value £10)20) (Table 7). Therefore, the

coverage of ESTs within the genes predicted from the WGS

assembly was very high. Conversely, 28 783/43 484 CDS

sequences (66%) aligned to one or more NCBI ESTs

(BLASTN e-value £10)20) (Table 6). The relatively low

proportion of predicted WGS CDS sequences that aligned

Distribution of mRNA length

Gene length (bp) (window = 50 bp)

Perc

ent o

f gen

es(%

)

0 3000 6000 9000 12 0000

1

2

3

4

5L.usitatissimumA.thalianaC.sativusG.maxP.trichocarpaR.communis

Distribution of CDS length

CDS length (bp) (window = 50 bp)

Perc

ent o

f gen

es(%

)

0 1000 2000 3000 4000 50000

3

6

9L.usitatissimumA.thalianaC.sativusG.maxP.trichocarpaR.communis

Distribution of exon length

Exon length (bp)(window = 10 bp)

Perc

ent o

f exo

ns(%

)

0 100 200 300 400 5000

3

6

9L.usitatissimumA.thalianaC.sativusG.maxP.trichocarpaR.communis

Distribution of intron length

Intron length (bp)(window = 10 bp)

Perc

ent o

f int

rons

(%)

0 100 200 300 400 5000

7

14

21

28L.usitatissimumA.thalianaC.sativusG.maxP.trichocarpaR.communis

(a) (b)

(c) (d)

Figure 2. Gene prediction.

Distribution of the length of (a) mRNA, (b) CDS, (c)

exon and (d) intron features within the complete

set of predicted genes for flax and five other

representative dicots.

466 Zhiwen Wang et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

to flax ESTs may indicate that the coverage of flax genes in

EST databases is incomplete and/or that a considerable

proportion of the predicted CDS sequences do not represent

components of real transcripts. Both possibilities were

examined in the subsequent analyses described below.

We compared the predicted WGS proteins with peptide

sequences in the NCBI databases, and separately with

peptide sequences from the well-characterized Arabidopsis

and poplar genomes (Tuskan et al., 2006; Swarbreck et al.,

2008). We observed that 89.5% (38 920/43 484) of flax WGS

proteins aligned to one or more proteins from the NCBI nr

protein database (http://www.ncbi.nlm.nih.gov/protein), and

nearly the same proportion aligned with Arabidopsis

(38 876/43 484; 89.4%) or poplar proteins (39 746/43 484;

91.4%; BLASTP e-value £10)5). Because sequences from

different species were not expected to be identical, a lower

level of stringency was used in these cross-species compar-

isons than was used in the comparisons of flax ESTs to flax

CDS sequences. Overall, the high proportion of predicted

proteins from the flax WGS that aligned with proteins from

Arabidopsis, poplar and the NCBI nr database indicates that

the majority of genes predicted in the flax WGS are

legitimate protein-coding sequences. We also made con-

verse comparisons to determine what proportion of Arabi-

dopsis and poplar proteins were represented by at least one

flax protein. We found that 86.0% (23 575/27 416) of Arabi-

dopsis proteins and 86.4% (35 135/40 688) of poplar proteins

aligned to one or more predicted flax proteins (BLASTP

e-value £10)5). This level of coverage is consistent with the

alignment of predicted poplar and Arabidopsis proteins

reported after sequencing of the poplar genome (Tuskan

et al., 2006).

Functional annotation of predicted proteins

The Pfam-A database provides profile hidden Markov mod-

els of over 13 672 conserved protein families (Finn et al.,

2010). Approximately 79% of known proteins contain one or

more Pfam domains. We used PfamScan/HMMer3 to iden-

tify Pfam domains within the predicted genes of the flax

WGS assembly (ftp://ftp.sanger.ac.uk/pub/databases/Pfam/

Tools/; Eddy, 2011). As shown in Table 7, 77% (33 459/

43 484) of the proteins in the flax WGS assembly contained

one or more Pfam domains (Pfam-A 26.0, e-value £10)5).

Almost all of the 5312 Pfam-A domains were found to occur

at the same frequency in the predicted flax genes as in genes

from either Arabidopsis or poplar (Figure 3). Only 47

domains showed significantly different abundance in a

comparison between Arabidopsis and flax, and 53 domains

were different in abundance between flax and poplar

(v2 adjusted FDR q-value <0.05; Table S2). Among these,

Table 7 Alignment of predicted WGS CDS with ESTs, predicted genes and domains of other species

QueryQuery(number) Subject Algorithm Parameters

Queries withmatches Percentage

NCBI Lus ESTs 286 852 WGS CDS BLASTN 1.00E-20 248 210 86.5NCBI Lus ESTs 239 220 WGS CDS BLASTN 1E-20; length >400 222 846 93.2WGS CDS 43 484 NCBI Lus ESTs BLASTN 1.00E-20 28 783 66.2WGS CDS 43 484 NCBI nr BLASTP 1.00E-05 38 920 89.5WGS CDS 43 484 Ptr CDS BLASTP 1.00E-05 39 746 91.4WGS CDS 43 484 Ath CDS BLASTP 1.00E-05 38 876 89.4Ptr CDS 40 688 WGS CDS BLASTP 1.00E-05 35 135 86.4Ath CDS 27 416 WGS CDS BLASTP 1.00E-05 23 575 86.0WGS CDS 43 484 Pfam-A HMMer3 1.00E-05 33 459 77.0

Lus, Linum usitatissimum; Ptr, Populus trichocarpa; Ath, Arabidopsis thaliana.

(a) (b)Figure 3. Abundance of Pfam-A domains in pre-

dicted proteins from flax and other whole-

genome sequences.

For each domain, the number of proteins with at

least one occurrence of the domain is plotted.

Domains that are significantly more abundant in

one species (v2 FDR q-value <0.05) are plotted as

triangles; domains with similar abundance in

each species (v2 FDR q-value ‡0.05) are plotted as

circles. Comparison of (a) flax with A. thaliana

and (b) flax with P. trichocarpa. Full details of the

domains and their frequencies are given in Table

S2.

Whole-genome shotgun assembly of flax 467

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

12 domains (excluding domains assumed to represent un-

masked transposable elements) were significantly more

abundant in flax than in at least one of either Arabidopsis or

poplar (Figure 4). Some of the notable Pfam domains that

were enriched in flax included HSP70 (heat shock protein 70,

PF00012), GRAS transcription factors (PF03514) and BSP

(basic secretory protein, PF04450), BSP is found in peptid-

ases involved in defense. Agglutinin domains (PF07468),

which are also presumably involved in defense, were found

in 17 predicted flax genes. This agglutinin domain is not

found in any known proteins of either Arabidopsis or poplar

(although they are present in a diverse range of other spe-

cies; Figure 4). Conversely, domains that were under-repre-

sented in the predicted flax genes included many domains of

unknown function (DUFs), some classes of leucine-rich

repeats, wall-associated kinases, F-box-associated domains

and transcription factors. The functional and ecological

relevance of these observations is currently being tested.

Analysis of sequence divergence in duplicated genes

Because the number of genes predicted in the flax WGS

assembly was higher than some other plant species (e.g.

A. thaliana), we analyzed the gene set for evidence of recent

genome duplication. We performed two separate analyses,

using as input either the capillary-sequenced flax ESTs from

the NCBI GenBank database or the predicted CDS sequences

from the WGS assembly (http://www.ncbi.nlm.nih.gov/

nucleotide/). We identified 3644 duplicate gene pairs among

LusPtrRcoMes

GmaCsaMdoAth

CpaVviSbiZma

OsaBdiCrePpa

BSPBarwin

Alginate lyaseAgglutinin

Self_Incomp_S1SCRL

HSP70GRAS

TransferaseDUF4409

BBEPPR_2

Oxidored_q1MP

DUF659DUF594DUF577DUF4371DUF4220

97786672356415471036152524231471129711819537222250388

Figure 4. Pfam-A domain frequency in flax and other species.

All domains that were more abundant (v2 FDR <0.05) among predicted proteins of flax compared to either A. thaliana or P. trichocarpa are shown (labels in bold).

A subset of the domains that were significantly less abundant in flax compared to either species is also shown. Additional species for which whole-genome

sequence is available are shown for comparison. The width of the colored region indicates the number of genes containing a given Pfam-A domain within each

species. A different scale is used for each domain, and the number to the right of each bar indicates the total number of genes represented by that bar. Redundant

occurrences of the same domain within the same gene are counted only once. BSP, basic secretory protein; Barwin, PF00967; alginate lyase, PF08787; agglutinin,

PF07468; Self_Incomp_S1, plant self-incompatibility protein S1 (PF05938); SCRL, plant self-incompatibility response protein (PF06876); HSP70, heat shock protein 70

(PF00012); GRAS, GRAS family transcription factor (PF03514); transferase, PF02458; DUF4409, domain of unknown function (PF14365); BBE, berberine and

berberine-like (PF08031); PPR_2, PPR repeat family (PF13041); Oxidoreq_q1, oxidoreductase (PF00361); MP, viral movement protein (PF01107); DUF659 (PF04937);

DUF577 (PF04510); DUF4371 (PF14291); DUF4220 (PF13968). Ath, Arabidopsis thaliana; Bdi, Brachypodium distachyon; Cpa, Carica papaya; Cre, Chlamydomonas

reinhardtii; Csa, Cucumis sativa; Gma, Glycine max; Lus, Linum usitatissimum; Mdo, Malus domestica; Mes, Manihot esculenta; Osa, Oryza sativa; Ppa,

Physcomitrella patens; Ptr, Populus trichocarpa; Rco, Ricinus communis; Sbi, Sorghum bicolor; Vvi, Vitis vinifera; Zma, Zea mays.

(a) (b) Figure 5. Rate of substitutions per synonymous

sites (Ks) within duplicated gene pairs from (a)

capillary-sequenced flax ESTs and (b) predicted

CDS sequences from the flax WGS assembly.

468 Zhiwen Wang et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

the flax ESTs and 9920 pairs within the WGS CDS sequences,

and analyzed their divergence as nucleotide substitutions

per synonymous site per year (Ks). In both datasets, a dis-

tinct peak in the Ks distribution was observed at Ks = 0.15

(Figure 5). Based on this modal Ks value, a time of diver-

gence of 5–9 MYA was inferred depending on whether a

synonymous mutation rate per base of 1.5 · 10)8 or

8.1 · 10)9 is assumed (Koch et al., 2000; Lynch and Conery,

2000).

DISCUSSION

Assembly quality

The pace of plant genome sequencing is accelerating, aided

by advances in instrumentation and software. The clone-

by-clone sequencing strategy used to sequence the Arabid-

opsis genome has been supplanted by whole-genome

shotgun (WGS) approaches. WGS de novo assemblies have

typically relied on at least some component of long

sequencing reads (e.g. from dideoxy or pyrosequencing),

rather than the shorter (but less costly) reads produced by

Illumina or SOLiD sequencers (Applied Biosystems). How-

ever, a WGS de novo assembly of date palm was recently

reported that was based entirely on 36–84 bp Illumina reads,

with an N50 scaffold size of 30 kb (Al-Dous et al., 2011). Here

we describe de novo WGS assembly of flax based exclu-

sively on short reads (Table 1). The descriptive statistics for

the flax WGS assembly (Table 2), with scaffold N50 = 693 kb,

85% genome coverage in scaffolds, and coverage of up to

97% of the gene space as defined by ESTs (Table 3), provide

further evidence of the utility of the short-read de novo WGS

approach in plants. The occurrence of transposable ele-

ments (Table 5), NUPTs (Figure S3) and most types of non-

coding RNA (ncRNA) (Table 6) was typical of other plant

genomes sequenced to date (Richly and Leister, 2004; Tus-

kan et al., 2006). As a whole, our analyses showed that the

coverage and accuracy of the assembly was high, especially

at the scale of individual genes (Tables 3 and 7), but with

some limitations regarding long-range accuracy of the

assembly (Table 4 and Figure S1). Further improvements in

assembly accuracy will require the use of additional sources

of data such as the physical maps recently published for CDC

Bethune (Ragupathy et al., 2011). Nevertheless, we conclude

that the short-read de novo WGS approach is a highly effi-

cient strategy for characterization of the nearly complete

gene space of flax.

Predicted genes and protein domains

The masked WGS assembly supported prediction of a

consensus set of 43 484 genes (Table 7). This gene num-

ber is comparable to what has been reported in soybean,

poplar, and rice, but is substantially larger than Arabid-

opsis, Brachypodium or cucumber (Velasco et al., 2010).

The length distribution of exons of predicted flax proteins

was consistent with other species, although flax intron and

mRNA length tended to be shorter than other species in

the comparison (Figure 2). The proportion of predicted flax

genes that aligned with Arabidopsis genes (89.4%), that

contain Pfam domains (77%), and that align with one or

more proteins in the NCBI nr database (89.5%) is high

(Table 7). Moreover, the distribution of conserved protein

domains in flax is nearly equivalent when compared to

either Arabidopsis or poplar (Figure 3), and 93.2% of

known ESTs of at least 400 bp align with high stringency

to the predicted CDS sequences. Together these data

support our conclusion that the predicted WGS genes are

highly accurate and that the WGS assembly (and sub-

sequent gene prediction) produced very good coverage of

the gene space of flax. Therefore, the relatively low cov-

erage of the predicted WGS flax genes by ESTs (66.2%) is

probably due primarily to incomplete gene representation

in the ESTs, rather than systematic errors in gene predic-

tion.

Only a few functional groups appeared to be significantly

over-represented in flax compared to other species (Fig-

ure 4 and Table S2). The increased in abundance of

domains that are often related to plant defense responses

(e.g. lectins and basic secretory proteins), and the

decreased abundance of leucine-rich repeat domain-con-

taining proteins, raise the possibility of additional biotic

stress response strategies in flax. The increased abundance

of two domains nominally associated with self-incompati-

bility (PF05938 and PF06876; Figure 4) is probably not

functionally relevant to pollination control in this self-

compatible species, as we note that one of these domains

(SCRL) is also highly represented in Arabidopsis.

Evolution of the flax genome

Analysis of the rates of divergence of duplicated genes

(Figure 5) indicated that a whole-genome duplication event

probably occurred 5–9 MYA in the lineage of L. usitatissi-

mum. The same Ks distribution was observed in both CDS

sequences predicted from the Illumina WGS shotgun

assembly (Figure 5b) and capillary-sequenced ESTs (Fig-

ure 5a), demonstrating that the distribution is not an artifact

of the sequencing or assembly technique used. A recent

whole-genome duplication is also consistent with the large

gene number predicted for flax, compared for example to

Arabidopsis (Table 7), the relatively high number of dupli-

cated genes (9920) identified, and with the numbers of

chromosomes reported in L. usitatissimum and related

species (Figure 6). L. usitatissimum and the closely related

L. bienne both have n = 15 chromosomes, while sister

clades containing species such as L. grandiflorum and

L. lewisii have n = 8 and n = 9 chromosomes, respectively.

Therefore, the evolution of L. usitatissimum probably

involved chromosome doubling followed by loss of one or

more chromosomes. Comparison of patterns of gene family

Whole-genome shotgun assembly of flax 469

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

expansion in L. usitatissimum and L. bienne and other

congeners is ongoing.

In conclusion, de novo assembly of exclusively short,

paired reads obtained by approximately 94· raw coverage of

the flax genome was an efficient method of obtaining near-

complete and highly accurate information about the gene

space of this crop. A limitation to this approach is the

accuracy of the long-range assembly, as demonstrated by

imperfect alignments to BAC sequences. Nevertheless, the

gene-scale data are sufficient for further investigations into

the function and evolution of this species.

EXPERIMENTAL PROCEDURES

Plant growth and DNA isolation

Seeds of the L. usitatissimum linseed cultivar CDC Bethune wereobtained from its breeder, Gordon Rowland (Department of PlantSciences, University of Saskatchewan, Saskatoon, Canada). Theseseeds were the F19 generation of the original cross of variety Nor-Man · genotype FP857 (Rowland et al., 2002). All generations wereself-pollinated, and the final eight generations were obtained bysingle-seed descent. Seeds were surface-sterilized and grown axe-nically in polycarbonate vessels. DNA was extracted from etiolatedseedlings using a variation of a published protocol (Dellaporta et al.,1983), in which the concentration of NaCl in the extraction bufferwas 1 M. Upon resuspension of DNA in TE buffer (50 mM Tris and10 mM EDTA, pH 8), 12 ll of 10 mg/ml RNase A was added, and themixture was allowed to incubate at 37�C for 1 h. Subsequently aphenol/chloroform/isoamyl alchohol (25:24:1) extraction was per-formed to remove impurities. DNA was precipitated with 0.1volumes of 3 M NaOAc and 0.6 volumes of isopropanol, washed in70% EtOH, resuspended in 400 ll TE (10:0.1), and subsequentlyre-precipitated with 0.1 volumes of 3 M NaOAc and two volumes95% EtOH. DNA was washed and resuspended in TE. Aliquots of thesame DNA preparation were used for genome sequencing andfosmid library construction.

Flow cytometry

DNA content measurements (Galbraith et al., 1983) were performedon ten CDC Bethune seedlings germinated from the same source ofseeds used for WGS and fosmid sequencing. The measurementswere performed using an Accuri C6 flow cytometer (Becton, Dicksonhttp://www.bdbiosciences.com), with propidium iodide/RNase asthe nuclear DNA stain (Bharathan et al., 1994). The nuclear DNA

contents were calibrated to an external standard, Raphanus sativacv. Saxa (Dolezel et al., 2007), with a DNA content of 1.11 pg/2Cnucleus. A single preparation of the standard was run five times toprovide a mean DNA fluorescence peak position that was then usedas the reference for the DNA content calculations.

Whole-genome shotgun sequencing and assembly

Seven paired-end sequencing libraries were constructed accordingto the manufacturer’s protocols (Illumina, http://www.illumina.com/)at BGI-Shenzen. The nominal insert sizes of the libraries were300 bp, 500 bp, 2 kb, 5 kb and 10 kb. For libraries with an insert sizebetween 200 and 500 bp, library preparation proceeded as follows:genomic DNA fragmentation, end repair, adapter ligation, sizeselection and PCR. For the longer (‡2 kb) mate-paired libraries, DNAcircularization, digestion of linear DNA, fragmentation of circular-ized DNA, and purification of biotinylated DNA were performedbefore adapter ligation. After the libraries were constructed, thetemplate DNA fragments of the constructed libraries were hybrid-ized to the surface of flow cells, amplified to form clusters, and thensequenced using an Illumina GAII genome analyzer.

Raw reads were filtered to remove fragments that contained>2% unknown bases or poly(A) structure, or for which >60% ofbases for the large insert-size library data and 40% of bases for theshort-insert data showed a quality score £7. Reads were alsoremoved if more than 10 bp aligned to the adapter sequence(allowing a mismatch of £3 bp). The filtered reads were assembledusing SOAPdenovo as previously described (Li et al., 2009). Theassembly comprised three stages. In the first stage, short-insertlibrary data were split into k-mers, a de Bruijn graph wasconstructed and simplified, and then the k-mer path was con-nected to generate the contig file. In the second stage, all theusable reads were re-aligned onto the contig sequences, then thenumber of shared paired-end relationships between each pair ofcontigs was calculated and used to identify consistent andconflicting paired-ends used to construct the scaffolds. Finally,the paired-end information was used to retrieve read pairs that hadone end mapped to a unique contig and the other located in a gapregion, and then a local assembly was performed for thesecollected reads to fill the gaps.

Fosmid library construction and sequencing

Fosmid and BAC libraries were constructed from flax variety CDCBethune, and selected clones were sequences as described previ-ously (Ragupathy et al., 2011; Roach et al., 2011). Fosmids weredeposited in NCBI GenBank under accessions HQ902252 andJN133299–JN133301.

Annotation of transposable elements and ncRNA

Putative transposable element regions of the flax genome wereidentified using REPEATMASKER version 3.3.0 (http://www.repeat-masker.org/), RMBlast was used as the search algorithm with aSmith–Waterman cut-off of 225 (this cut-off was used for allRepeatMasker analyses), and a database with annotated de novorepeats was combined with the Viridiplantae database of trans-posable elements (update 20110920) for use as a library for com-parison to the shotgun assembly. To annotate the masked bases intheir respective transposable element superfamilies, a custom Perlscript was used (kindly provided by Robert Hubley, Institute forSystems Biology, Seattle, WA). De novo identification of transpos-able elements was performed using RepeatScout (Price et al., 2005),PILER (Edgar and Myers, 2005), LTR_finder (Xu and Wang, 2007) andLTR_STRUC (McCarthy and McDonald, 2003). RepeatScout was

Figure 6. Phylogram of flax (L. usitatissimum) and related species showing

haploid chromosome number (in black or shaded circle).

The star indicates the approximate placement of the whole-genome duplica-

tion that we infer occurred in the common ancestor of L. bienne and

L. usitatissimum. The tree and dated nodes are based on data from McDill

et al. (2009).

470 Zhiwen Wang et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 72, 461–473

used under the default parameters. Repeats identified by Repeat-Scout were filtered for low complexity using tandem repeats finder(Benson, 1999) and nseg (Wootton and Federhen, 1993), and thefiltered library was used to mask the flax genome. Repeats withfewer than ten hits in the genome were eliminated from the library.For PILER-DF analysis, the full genome was compared to itself usingPALS (part of the PILER implementation) with default parameters.Families of dispersed repeats were created using a minimum familysize of three members and a maximum length difference of 5%between all family members. The consensus sequence for eachfamily was created after aligning the sequences using MUSCLE

(Edgar, 2004). LTR transposable elements were found usingLTR_finder with option –w2 to obtain a table output that was parsedto obtain the sequences corresponding to the elements.LTR_STRUC (McCarthy and McDonald, 2003) was used underdefault parameters.

Four types of non-coding RNAs were detected by searching theunmasked scaffold assemblies using tRNASCAN-SE (version 1.23)(Lowe and Eddy, 1997), and BLAST alignment with RFAM database(release 9.1) (Griffiths-Jones et al., 2003) and Arabidopsis thalianaand Oryza sativa full-length rRNAs, followed by INFERNAL (version0.81) (Nawrocki et al., 2009).

Gene prediction and annotation

Gene prediction was performed using a combination of de novo andhomology-based methods. De novo predictions were performed onthe repeat-masked WGS assembly using AUGUSTUS (version 2.5.5)(Stanke et al., 2008) and GLIMMERHMM (version 3.0.1) (Majoroset al., 2004). Flax ESTs were also aligned to the WGS assembly usingBLAT (blat-34, identity ‡0.98, coverage ‡0.98) to generate splicedalignments, which were linked according to their overlap using PASA

(Haas et al., 2003). Plant proteins of other species were also mappedto the WGS assembly using TBLASTN (Blastall 2.2.23, Altschul et al.,1997) with an e-value cut-off of 10)5. The aligned sequences as wellas their query proteins were then filtered and passed to GENEWISE

(version 2.2) (Birney et al., 2004). Gene models from de novo pre-dictions and EST and protein alignments were integrated usingGLEAN (Elsik et al., 2007) to produce a consensus gene set.

Duplicate gene pair analysis

All analyses were performed independently on two datasets. First,we downloaded all 286 852 ESTs available for L. usitatissimum inNovember 2011 from the NCBI database (http://www.ncbi.nlm.nih.gov/nucleotide/) and assembled them into unigenes using CAP3

with default settings (Huang and Madan, 1999). Second, we used all43 484 predicted CDSs from the flax genome project.

Analyses were performed with the DupPipe pipeline (Barkeret al., 2008) using default parameters. In short, sequences wereclustered into gene family members by parsing the results of adiscontiguous MegaBlast (Zhang et al., 2000; Ma et al., 2002) toretain those that showed at least 40% sequence similarity over aminimum of 300 bp. Reading frames for each sequence pair wereidentified by comparison with all available plant protein sequencesof GenBank (Wheeler et al., 2007) using BLASTX (Altschul et al.,1997). Best-hit proteins were then paired with each nucleotidesequence at a minimum cut-off of 30% sequence similarity over atleast 150 sites. Sequences that did not meet these criteria wereremoved from further analyses. Each gene was aligned against itsbest-hit protein using GENEWISE 2.2.2 (Birney et al., 2004) to deter-mine the reading frame. Amino acid sequences for each gene wereestimated by using the highest scoring Genewise DNA–proteinalignments. Amino acid sequences for each duplicate pair werethen aligned using MUSCLE 3.6 (Edgar, 2004), and used to align their

corresponding DNA sequences using REVTRANS 1.4 (Wernerssonand Pedersen, 2003). Ks values (synonymous substitution rates) foreach duplicate pair were calculated using the maximum-likelihoodmethod implemented in codeml of the PAML package (Yang, 1997)under the F3-4 model (Goldman and Yang, 1994). Further cleaningof the dataset was then performed to remove duplication eventsthat may bias the results. To reduce the possibility that identicalgenes were represented in the dataset, but were missed by TGICLclustering (Pertea et al., 2003) due to alternative splicing, all Ks

values from one member of a duplicate pair with Ks = 0 wereremoved. Further, to reduce the multiplicative effects of multi-copygene families on Ks values, simple hierarchical clustering was usedto construct phylogenies for each gene family, identified as single-linked clusters, and the nodal Ks values were calculated.

Data access

WGS assembly data were deposited at NCBI GenBank underGenomeProject ID #68161 and Sequence Read Archive accessionSRA038451. Fosmid and BAC sequences were submitted to Gen-Bank as accessions HQ902252, JN133299–JN133301 and JX174444–JX174449. Additional resources, bulk data downloads and aGbrowse (Stein et al., 2002) implementation are available at http://www.linum.ca and http://phytozome.org.

ACKNOWLEDGEMENTS

Funding was provided by Genome Alberta/Genome Canada, theGovernment of Alberta, Alberta Innovates Technology Futures-iCORE, Institut National de la Recherche Agronomique France andthe Indian Council of Agricultural Research.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Sequencing depth distribution.Figure S2. Diagrams showing alignment of BACs and fosmids withflax WGS scaffolds.Figure S3. Size distribution of putative NUPT regions in the flaxWGS assembly.Table S1. List of putative miRNAs identified in the flax WGSassembly.Table S2. Representation of Pfam-A domains in predicted genes ofthe flax WGS assembly and in genomes of other selected species.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but is not copy-edited or typeset. Technical support issuesarising from supporting information (other than missing files)should be addressed to the authors.

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